Fusion proteins containing glutathione-s-transferase

Abstract

A recombinant DNA molecule comprising a nucleotide sequence which codes for expression of a fusion protein in which a foreign protein or peptide is fused with the enzyme glutathione-S-transferase, is disclosed, as well as expression vectors or host cells containing such a molecule. Optionally, the foreign protein or peptide is fused to the enzyme through a cleavable link. Also disclosed is an expression vector having inserted therein a nucleotide sequence capable of being expressed as the enzyme glutathione-S-transferase followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign protein or peptide fused to the glutathione-S-transferase.

I claim: 
     
       1. A recombinant DNA molecule comprising a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase, and wherein said fusion protein is capable of binding to glutathione. 
     
     
       2. The recombinant DNA molecule according to claim 1, wherein said glutathione-S-transferase is the 26kDa glutathione-S-transferase of Schistosoma japonicum. 
     
     
       3. The recombinant DNA molecule according to claim 1 wherein said second protein or peptide is fused to said glutathione-S-transferase through a cleavable link. 
     
     
       4. The recombinant DNA molecule according to claim 3, wherein said cleavable link is cleavable by a site specific protease. 
     
     
       5. The recombinant DNA molecule according to claim 4, wherein said clearable link is cleavable by thrombin, blood coagulation factor X a  or renin. 
     
     
       6. The recombinant DNA molecule of claim 1 wherein said glutathione-S-transferase is a mammalian glutathione-S-transferase. 
     
     
       7. An expression vector comprising the recombinant DNA molecule of any one of claims 1, 2-5 and 6. 
     
     
       8. A host cell comprising the expression vector of claim 7. 
     
     
       9. The host cell of claim 8 wherein said host cell is a bacterial cell. 
     
     
       10. The host cell according to claim 9 which is E. coli. 
     
     
       11. An expression vector comprising, in the 5' to 3' direction, a promoter, a nucleotide sequence encoding glutathione-S-transferase and a nucleotide sequence comprising at least one restriction endonuclease recognition site. 
     
     
       12. The expression vector according to claim 11, wherein said glutathione-S-transferase is the 26kDa glutathione-S-transferase of Schistosoma japonicum. 
     
     
       13. The expression vector according to claim 11, which comprises a plasmid selected from the group consisting of pGEX-1, pSj10 DBamI and pSj10 DBam7Stop7. 
     
     
       14. An expression vector comprising, in the 5' to 3' direction, a promoter, a nucleotide sequence encoding glutathione-S-transferase, a nucleotide sequence encoding a clearable link and a nucleotide sequence comprising at least one restriction endonuclease recognition site. 
     
     
       15. The expression vector according to claim 14, wherein said cleavable link is cleavable by a site specific protease. 
     
     
       16. The expression vector according to claim 14 which is pGEX-2T or pGEX-3X. 
     
     
       17. The plasmid pGEX-1. 
     
     
       18. The plasmid pGEX-2T. 
     
     
       19. The plasmid pGEX-3X. 
     
     
       20. A method of producing a fusion protein comprising glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase which comprises: (a) transforming a host cell with an expression vector comprising a promoter operatively linked to a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of glutathione-S-transferase and wherein said fusion protein is capable of binding to glutathione;   (b) culturing said host cell under conditions such that said fusion protein is expressed in recoverable quantity;   (c) lysing said host cell; and   (d) purifying said fusion protein by glutathione-affinity chromatography.   
     
     
       21. A method of producing a protein or peptide which comprises: (a) transforming a host cell with an expression vector comprising a promoter operatively linked to a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises glutathione-S-transferase and a second protein or peptide fused with the COOH-terminus of glutathione-S-transferase through a cleavable link wherein said fusion protein is capable of binding to glutathione;   (b) culturing said host cell under conditions such that said fusion protein is expressed in recoverable quantity;   (c) lysing said host cell;   (d) purifying said fusion protein by glutathione-affinity chromatography;   (e) cleaving said protein or said peptide from said glutathione-S-transferase; and   (f) isolating said protein or said peptide.   
     
     
       22. A fusion protein comprising glutathione-S-transferase and a second protein or peptide fused directly or indirectly with the COOH-terminus of said glutathione-S-transferase wherein said fusion protein is capable of binding to glutathione. 
     
     
       23. A fusion protein comprising from amino to carboxyl terminus, glutathione-S-transferase, a cleavable linker and a second protein or peptide wherein said fusion protein is capable of binding to glutathione. 
     
     
       24. The fusion protein of claim 23 wherein said cleavable linker is cleavable by a site-specific protease. 
     
     
       25. The fusion protein of claim 24 wherein said protease is selected from the group consisting of thrombin, blood coagulation Factor Xa and renin.